DNA interstrand crosslinks (ICLs) are cytotoxic DNA lesions that covalently link the two strands of DNA and block DNA replication and RNA transcription. ICLs are the primary lesion generated by several of the most commonly used chemotherapeutic agents, including cisplatin and bi-functional alkylating agents. Thus, the efficacy of these agents depends on the efficiency with which ICLs are repaired, and better understanding of how cells repair ICLs may lead to improved chemotherapeutic agents and/or protocols. Nevertheless, the molecular mechanism(s) of ICL repair in human cells remains poorly understood. The research proposed here will study the molecular mechanism(s) of ICL repair in human cells using a novel in vitro assay for ICL repair. This assay uses a defined substrate DNA with a single psoralen ICL and the SV40 origin of replication. Preliminary results indicate that repair of the ICL in this substrate by human proteins is coupled to DNA replication, and requires BRCA2. Previous studies suggest that ICL repair in human cells is accomplished in the following four kinetically distinct steps: (1) unhooking of the ICL on one strand and induction of a DNA replication dependent double-strand break (DSB), (2) translesion DNA synthesis (TLS) across from the unhooked ICL, (3) processing of the DSB and restoration of the stalled DNA replication fork, and (4) removal of the residual unhooked ICL. The goal of this proposal is to define the human protein factors required to accomplish ICL repair and to define the roles played by these proteins in ICL repair using the in vitro ICL repair assay. The specific aims of the proposal are: 1) Define the role of structure-specific endonuclease complexes XPF/ERCC1 and MUS81/MMS4 in human ICL repair;2) Define the roles of TLS polymerases PolQ and PolN in ICL repair using purified His-tagged human proteins and a gapped DNA substrate with an unhooked psoralen ICL;3) Define the role of DSB repair proteins in ICL repair with particular focus on BRCA2 and Fanconi anemia (FA) proteins, whose inactivation confers hypersensitivity to DNA crosslinking agents. The experiments proposed here will use RNAi to analyze the proteins required for each step of ICL repair. ICL repair assays will be carried out with extracts from RNAi-treated human cells, and the role of the knocked-down protein will be deduced by analyzing the structure and amount of the reaction products or intermediates that accumulate in the ICL repair reaction. Results with knocked down extracts will also be confirmed by adding back purified proteins. These studies will contribute to our basic understanding of human ICL repair and lay the groundwork for reconstituting ICL repair in vitro using purified repair proteins. PUBLIC HEALTH RELEVANCE: Defects in DNA interstrand crosslink (ICL) repair lead to genetic instability in human cells and cause rare genetic diseases such as familial breast cancer and Fanconi anemia (FA). This research proposed here will identify and characterize human protein components involved in using a novel cell-free ICL repair assay. The results of the proposed studies will improve our understanding of the molecular mechanism of ICL repair in human cells. In addition, the proposed experiments will also investigate structure-function relationships in BRCA2 and decipher the function of FA proteins in ICL repair. These studies will give new insights into understanding tumor suppressor functions of BRCA2 and FA proteins.